超顺磁性氧化铁标记西藏小型猪骨髓间充质干细胞体内示踪的实验研究

发布时间：2019-05-19 23:38

【摘要】：第一部分应用新型超顺磁性氧化铁体外标记骨髓间充质干细胞目的:探讨新型超顺磁性氧化铁(new superparamagnetic iron oxide,SPIO)粒子体外标记骨髓间充质干细胞(Bone mesenchymal stem cell ,BMSCs)的安全性和可行性。方法:密度梯度离心法分离西藏小型猪BMSCs,体外扩增、培养和鉴定。应用不同浓度的SPIO对BMSCs进行体外标记,应用普鲁士蓝染色评价标记效率;透射电镜观察细胞内摄取的SPIO;四唑盐(MTT)比色实验评价标记后细胞生长情况;台盼蓝拒染实验观察标记后细胞的活性;碘化丙啶(PI)染色法检测不同浓度SPIO对细胞周期的影响,成骨、成脂诱导鉴定标记后的BMSCs的分化潜能。选取SPIO标记的BMSCs行MR成像,测量不同扫描序列标记细胞管信号强度的改变。结果:BMSCs经SPIO标记后,普鲁士蓝染色观察SPIO标记阳性率近100%,透射电镜观察可见细胞浆内有多少不等铁颗粒;浓度为25ug/mL的新型SPIO标记24h,对BMSCs的活性、生长周期和分化潜能无明显影响。标记的BMSCs在T2WI尤其是GRE(T2* WI)序列随着细胞数量的增加信号改变越明显。结论:在体外SPIO可有效标记BMSCs,适当浓度的SPIO标记对BMSCs的生物学活性没有明显影响,在MRT2*WI序列可敏感显像标记的BMSCs。第二部分生理状态下骨髓间充质干细胞在西藏小型猪体内示踪的研究目的:观察生理状态下经不同途径输注SPIO标记的西藏小型猪BMSCs在体内的分布情况。方法:SPIO标记西藏小型猪BMSCs。分别经外周静脉和肾动脉输注已标记的BMSCs(2×10~6/Kg),输注后当天行磁共振扫描。24h后分别取心脏、肝脏、脾脏、肺脏、胰腺及双侧肾脏的病理标本。组织切片行普鲁士蓝染色及H-E染色观察阳性细胞在各脏器中的分布。结果:西藏小型猪经外周静脉及肾动脉输注SPIO标记的BMSCs在磁共振成像上未见明显显影。病理组织学检查结果显示经外周静脉输注后,在肺脏内发现BMSCs;经肾动脉输注后在肺内及输注一侧肾脏发现BMSCs。结论:西藏小型猪在生理状态下经外周静脉输注SPIO标记的BMSCs主要分布于肺脏,经肾动脉输注SPIO标记的BMSCs主要分布于肺脏及输注侧肾脏。第三部分肾移植术后骨髓间充质干细胞在西藏小型猪体内示踪的研究目的:观察肾移植术后经不同途径输注经SPIO标记的BMSCs在西藏小型猪体内的分布情况,探讨不同输注途径对BMSCs分布的影响。方法:建立西藏小型猪肾移植受体动物模型,分别经移植肾动脉和外周静脉输注SPIO标记的BMSCs (2×10~6/Kg)。24h后分别取心脏、肝脏、脾脏、肺脏、胰腺、自体肾脏及移植肾等标本。组织切片行普鲁士蓝染色及H-E染色,观察SPIO标记的BMSCs在各脏器中的分布。结果:经外周静脉输注SPIO标记的BMSCs后,仅在肺组织发现BMSCs;而经移植肾动脉输注后,则在移植肾及肺组织内均发现BMSCs,且多位于皮质区肾小球内。输注相同数量的BMSCs,经移植肾动脉输注与经外周静脉输注相比较,肺组织内BMSCs更少,差异有统计学意义(P0.05). 结论:肾移植术后,经外周静脉输注后BMSCs主要分布于肺脏,经移植肾动脉输注后BMSCs主要分布于移植肾及肺脏,并且在肺组织中BMSCs的数量有差异。
[Abstract]:The first part uses new superparamagnetic iron oxide to mark the bone marrow mesenchymal stem cells in vitro Objective: To investigate the safety and feasibility of novel superparamagnetic iron oxide (SPIO) particles in the in vitro labeling of bone marrow mesenchymal stem cells (BMSCs). Methods: BMSCs from Tibetan small pigs were isolated by density gradient centrifugation, and cultured in vitro. And the cell growth of the labeled cells was evaluated by means of a transmission electron microscope; the cell growth after the labeling was evaluated by a four-salt (mtt) colorimetric assay; and the labeled cells were observed by trypan blue rejection. The effects of different concentrations of SPIO on the cell cycle, osteogenesis and lipogenesis-induced differentiation of BMSCs were detected by the method of PI. Potential. BMSCs with SPIO markers are selected for MR imaging, and the signal intensity of cell tubes labeled with different scanning sequences is measured. Results: After the BMSCs were labeled by SPIO, the positive rate of SPIO was nearly 100% in the staining of Prussian blue, and the number of non-iron particles in the cytoplasm of BMSCs was observed by transmission electron microscope. The new type of SPIO with a concentration of 25 ug/ mL for 24 h, the activity, the growth cycle and the differentiation potential of BMSCs were observed. No significant effect. The labeled BMSCs on T2WI, especially the GRE (T2 * WI) sequence, increased with the number of cells Conclusion: In vitro SPIO can be used to mark BMSCs, and the SPIO markers of appropriate concentration have no significant effect on the biological activity of BMSCs. The BMSCs of the second part of the BMSCs in Tibet The purpose of the study on the in vivo tracing of small pigs: to observe the BM of the small pigs with SPIO markers through different routes under physiological conditions The distribution of SCs in the body. Method: SPIO BMSCs were infused into the small pigs of Tibet. The labeled BMSCs (2-10-6/ Kg) were transfused with the peripheral veins and the renal arteries. The heart, the liver, the spleen, the lung and the pancreas were taken after 24 h. The pathological specimens of the adenoid and bilateral kidneys. The tissue sections were stained with Prussian blue and stained with H-E. The distribution of SPIO-labeled BMSCs by peripheral vein and renal artery infusion of small-sized pigs in Tibet. No significant development was seen on magnetic resonance imaging. The results of the histological examination showed that BMSCs were found in the lung after the peripheral vein infusion, and in the lungs after the renal artery infusion. BMSCs were found in the kidney of one side of the infusion. Conclusion: The BMSCs marked by SPIO in the physiological state of the small pigs in Tibet are mainly distributed in the lung, and the SPIO-marked BMS is infused through the renal artery. Cs are mainly distributed in the lung and the kidney of the infusion side. The third part of the bone marrow after renal transplantation The purpose of this study was to observe the distribution of the SPIO-labeled BMSCs in the small pigs of Tibet after renal transplantation. The effect of different infusion methods on the distribution of BMSCs was discussed. Methods: The model of small pig kidney transplantation recipients in Tibet was established, and the BMSCs (2-10-6/ Kg) were respectively infused with SPIO-labeled BMSCs (2-10-6/ Kg). The heart, liver and spleen were respectively taken after 24 h. The tissue sections were stained with Prussian blue and H-E, and S was observed by the staining of Prussian blue and H-E. The results showed that BMSCs were only found in lung tissue after peripheral vein infusion of SPIO-labeled BMSCs. BMSCs were found and located in the glomerulus of the cortical area. The same number of BMSCs were infused, and the transplanated renal artery infusion was compared with the transvenous infusion and the BM in the lung tissue Conclusion: BMSCs are mainly distributed in the lung after renal transplantation, and BMSCs are mainly distributed in the transplantation after renal transplantation.
【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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